Gas-encapsulated acoustically responsive stabilized microbubbles and methods for treating cardiovascular disease

ABSTRACT

Acoustically responsive stabilized microbubbles formulated with a phospholipid monolayer shell, an encapsulated bioactive gas, and an encapsulated perfluorocarbon gas of the formula C x F y  in a volume ratio of from about 10:1 to about 1:10, wherein X is greater than or equal to 3, are disclosed. Also provided are methods for promoting localized vasodilation in a patient in need thereof by delivering a microbubble comprising a phospholipid monolayer shell and an encapsulated bioactive gas locally to a target diseased section of the patient&#39;s vasculature; and releasing the bioactive gas at the target diseased section, wherein the microbubble comprises the bioactive gas in a ratio of from about 10:1 to about 1:10 by volume with a perfluorocarbon gas.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/788,224, filed Oct. 19, 2017, which is a continuation-in-part of U.S.application Ser. No. 14/957,705, filed Dec. 3, 2015, and claims benefitunder 35 U.S.C. § 119(e) to U.S. Provisional Application Ser. No.62/086,749, filed Dec. 3, 2014, the entire disclosures of which areincorporated herein by this reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under R01 HL074002awarded by the National Institute of Health. The Government has certainrights in the invention.

TECHNICAL FIELD

The subject matter of this application relates to gas-encapsulatedacoustically responsive stabilized microbubbles and methods forpromoting localized vasodilation in patients in need thereof.Specifically, the acoustically responsive microbubbles disclosed hereinare useful in the treatment of diseases and conditions that benefit frompromoting vasodilation, including cardiovascular disease, infectiveendocarditis, cerebrovascular disease, stroke, myocardial infarction,and the like.

BACKGROUND

Many diseases and conditions benefit from therapeutic promotion ofvasodilation. Among these, cardiovascular disease (CVD) is currently theleading cause of death and is predicted to be the number one cause ofdisability worldwide by 2030 (World Health Organization 2014). In theUnited States, approximately 1 of every 4 deaths from 1999-2015 was dueto CVD (Centers for Disease Control and Prevention, 2016). Ischemicheart disease and stroke were the leading primary causes of prematuredeath. The overall economic impact of CVD was estimated to be $200billion annually in the U.S., and is expected to increase in futuredecades. More effective diagnostic tools and therapies are necessary tolimit the growing burden of CVD in the U.S. and worldwide, particularlythe diseases which manifest in unwanted clotting within the arteries ofthe heart or brain.

A major contributor to acute cardiovascular events and sudden deaths isthe development of atherosclerotic plaques, a progressive thickening ofthe arterial wall due to the accumulation of cholesterol. Rupture ofatherosclerotic plaques can form thrombi that occlude blood flow,potentially leading to a life-threatening event. Thrombi occurring inthe coronary artery can lead to a heart attack, and in cerebral arteriescan lead to ischemic stroke.

The early detection and treatment of CVD is vital to assess the risk ofvulnerable plaques leading to an acute cardiovascular event. However,screening for vulnerable atherosclerotic plaque using current imagingmodalities poses specific challenges. Direct visualization usingnoninvasive imaging methods, e.g. carotid ultrasound, cardiovascularcomputed tomography, magnetic resonance imaging, and positron emissiontomography, are preferable for early diagnosis of vulnerableatherosclerotic plaque in high-risk patients. For example, carotidultrasound with measurement of the intima-media thickness within theartery wall offers a way to diagnose the extent of subclinicalatherosclerotic vascular disease, assess risk, and may offer a means toidentify disease progression and monitor the effectiveness of preventivetherapies. The use of microbubble based ultrasound contrast agents as acomplementary tool to enhance vascular ultrasound imaging, known ascontrast-enhanced ultrasound imaging, is emerging as an important methodin facilitating the detection and characterization of atheroscleroticdisease.

The use of microbubbles as ultrasound contrast agents (UCAs) in vascularimaging is well established. Most commercially available UCAs consist ofgas-filled microbubbles which have mean diameters between 1-5 μm and areencapsulated with a protein, polymer, or lipid shell. Albunex® (GEHealthcare) was the first UCA approved by the U.S. Food and DrugAdministration and consisted of an air-filled microbubble encapsulatedby an albumin shell. Second generation UCAs such as Optison® (GEHealthcare), Definity® (Lantheus Medical Imaging) and Lumason® (BraccoDiagnostics, Inc.) contain high-molecular-weight gases (e.g. C₃F₈ andSF₆ respectively), which have lower solubility in blood and thusincrease the lifetime of the microbubbles in circulation. The lowdensity and high compressibility of the gas core in UCAs enablesefficient ultrasound scattering. Thus, the injected agents areacoustically responsive, or echogenic, and function as intravasculartracers which can be visualized using ultrasound.

In addition to traditional contrast-enhanced ultrasound imaging, therehas been recent interest in advancing the applications of UCAs formolecular imaging of atherosclerosis. Molecular imaging techniques withtargeted UCAs are being used increasingly for noninvasive diagnosis ofinflammation, thrombus, and neovascularization. Targeted microbubbleagents are also being developed for controlled drug-deliveryapplications and have been vigorously promoted for therapeuticapplications in the treatment of CVD. Targeted UCAs are functionalizedby engineering the gas-encapsulating shell to contain molecules thatadhere to cells, which express disease-specific markers (e.g.,aminoacids) on the membrane. Phospholipid-shelled UCAs are of particularinterest for this purpose, because they can be targeted to molecularcomponents of disease by attaching specific ligands to the surface.

Phospholipid-shelled UCAs represent one type of UCA that is currentlyavailable for clinical use. The lipid molecules employed in theformulations are typically amphiphilic molecules which spontaneouslyform micelle structures that can encapsulate a gas microbubble in anaqueous environment. The lipids are surface-active molecules(surfactants) that orient their hydrophilic polar groups outside towardsthe surrounding aqueous medium and their hydrophobic tails inside awayfrom the water, stabilizing the microbubble and largely preventing thegas from escaping the encapsulation. Lipid-based ultrasound contrastagents such as Definity® and Lumason® (which was recently approved forclinical use in the U.S. but has been marketed as SonoVue® in Europe andAsia since 2001) are commercially available for diagnostic applications.MicroMarker® (VisualSonics, Toronto, Canada; Bracco Research SA, Geneva,Switzerland) and Targestar® (Targeson Inc., San Diego, Calif., USA) areexamples of targeted phospholipid-shelled UCAs currently available forpre-clinical investigational use.

A more recent formulation in the broad category of phospholipid-shelledUCAs, known as echogenic liposomes (ELIP), has been developed whichencapsulates both a gas and an aqueous phase (Alkan-Onyuksel et al.1996; Huang et al. 2001). Standard liposomes are characterized by aphospholipid bilayer shell, which encapsulates an aqueous compartment.ELIP are said to be echogenic because they contain a gas microbubblethat is highly reflective to ultrasound waves at low intensities. Theexact location of the entrapped gas pockets in ELIP has not been fullyascertained, and may be due to gas pockets stabilized by lipidmonolayers within the liposome, or within the lipid bilayer shell.

Targetable drug-delivery systems represent a fast developing area ofnanotechnology and are expected to have a dramatic impact on medicine inthe future. Many nano-scale drug carriers, such as liposomes, micelles,and polymer nanocapsules, have been developed or are under developmentfor encapsulation and delivery of therapeutic drugs. Liposomes are aconvenient, biologically compatible vehicle for administration of poorlysoluble drugs, and are among the first generation of nano-scale drugdelivery systems to be approved for clinical use and known asnanomedicines (Moghimi et al. 2005).

Gregoriadis and Ryman (1971) were the first to report on the use ofliposomes as drug carriers for directed delivery. The authorshypothesized that encapsulation of enzymes within the aqueous innercompartment of liposomes would aid in directing the payload to aparticular tissue and alleviate some of the problems associated withimmunological response to the proteins in circulation. They found thatliposomes remain largely intact during circulation and are cleared bylysosomes in the liver (and to a lesser extent in the spleen). Sincethen, liposome based drug-delivery systems have been developed usingchemotherapeutic agents for cancer therapy, thromolytic agents, andgenes, in addition to enzymes.

Most of the currently approved liposome formulations represent a basicform of nanomedicine involving a passive targeting and drug releaseprocess known as the enhanced permeability and retention (EPR) effect.This approach relies on extravasation and accumulation of theliposome-encapsulated drug at the target site, and is particularlysuited for cancer therapy applications due to the enhanced vascularpermeability of tumors compared with normal tissue. Because tumors arehighly vascularized and often lack effective lymphatic drainage,liposomes tend to accumulate in tumors much more than they do in normaltissues, resulting in increased drug uptake in these regions. AlthoughEPR is a rudimentary passive targeting method, it is a key reasonliposomes are currently the most widely used drug nanocarrier in cancertherapy. To realize the drug delivery potential of liposomes for otherapplications fully, however, it is important to develop agents with anactive triggering mechanism that allows the drug to be delivered in amore controlled fashion. Echogenic microbubbles, by virtue of theirability to encapsulate gas as well as therapeutic drugs, offer such apossibility.

Recently, ultrasound has been investigated as a method to triggerenhanced drug delivery within the human vasculature. The potential ofultrasound to control drug delivery spatially and temporally in anon-invasive manner is broadly appealing. Ultrasound-mediated drugdelivery (UMDD) has been demonstrated in a number of tissue beds, forexample the blood-brain barrier, cardiac tissue, prostate, and largearteries.

Acoustic cavitation is one physical mechanism that is hypothesized toinfluence UMDD. Cavitation as used herein refers to linear or nonlinearbubble activity that can occur near vessel walls within the vasculatureupon ultrasound exposure, which can exert mechanical stress on nearbycells and junctions. Mechanical stress can disturb the barriers to drugdelivery such as endothelial tight junctions or phospholipid membranes,via transient permeabilization. In vivo, cavitation can be nucleated atmoderate acoustic pressure amplitudes (<0.5 MPa) by ultrasound contrastagents (UCAs).

Nitric Oxide (NO) is a gas molecule that dynamically modulates thephysiological functions of the cardiovascular system, which includerelaxation of vascular smooth muscle, inhibition of plateletaggregation, and regulation of immune responses. Because a reduced NOlevel has been implicated in the onset and progression of variousdisease states, NO is expected to provide therapeutic benefits in thetreatment of cardiovascular diseases, such as essential hypertension,stroke, coronary artery disease, atherosclerosis, platelet aggregationafter percutaneous transluminal coronary angioplasty, andischemia/reperfusion injury. To date, pharmacologically active compoundsthat can release NO within the body, such as organic nitrates and sodiumnitroprusside, have been used as therapeutic agents, but their efficacyis significantly limited by their rapid NO release, poor distribution tothe target site, toxicity, and induction of tolerance. Attenuation ofnitric oxide production in the etiology of atherosclerosis progressionand diabetic vascular disease further highlights the need for noveltherapeutic nitric oxide modulation and delivery strategies. Effectivedelivery of bioactive NO to target cardiovascular tissue remains acompelling need in the art.

Xenon is a gas molecule that induces robust cardioprotection andneuroprotection through a variety of mechanisms. Through its influenceon Ca²⁺, K⁺, KATP/HIF, and NMDA antagonism, xenon is neuroprotectivewhen administered before, during and after ischemic insults. Xenon hasparticular promise as a bioactive agent for the treatment ofcerebrovascular diseases and conditions. However, effective delivery ofbioactive xenon to target cardiovascular or cerebrovascular tissueremains a need in the art.

Hydrogen sulfide is an endogenously produced gasotransmitter involved inthe regulation of nervous system, cardiovascular functions, inflammatoryresponse, gastrointestinal system, and renal function. Hydrogen sulfidegas has therapeutic potential for diseases such as arterial andpulmonary hypertension, atherosclerosis, ischemia-reperfusion injury,heart failure, peptic ulcer disease, acute and chronic inflammatorydiseases, Parkinson's and Alzheimer's disease, and erectile dysfunction.Effective delivery of bioactive hydrogen sulfide to target diseasedsections of vasculature remains a need in the art.

Hence, the need exists for improved compositions and methods fortargeted ultrasound-mediated delivery of therapeutic bioactive gases.

SUMMARY

Accordingly, the instant disclosure provides novel bioactive gas-loadedacoustically responsive stabilized microbubbles designed to exploit thebenefits discovered upon intensive investigation of the properties andrelease profiles of microbubbles comprising various gases, combinationsof gases, and ratios of specific gas combinations. Acousticallyresponsive stabilized microbubbles were formulated and properties andrelease profiles characterized to provide compositions and methods ofpromoting vasodilation in a patient that exhibit superior and unexpectedbenefits in the delivery and safe release of bioactive gases frommicrobubbles at target diseased vasculature sites. The stabilizedmicrobubbles provide both diagnostic and therapeutic benefit.

One embodiment provides a method for promoting vasodilation in a patientin need thereof. The method comprises: delivering a stabilizedmicrobubble comprising a phospholipid monolayer shell and anencapsulated bioactive gas locally to a target diseased section of thepatient's vasculature; and releasing the bioactive gas at the targetdiseased section, wherein the microbubble comprises the bioactive gas ina volume ratio of from about 10:1 to about 1:10 with a perfluorocarbongas.

Another embodiment is directed to a method of manufacturing anacoustically responsive microbubble effective for ultrasound-mediateddrug delivery, the method comprising: providing a lipid dispersioncomprising phospholipids in a sealed receptacle, evacuating air from aheadspace of the sealed receptacle, injecting a volume ratio of abioactive gas and a perfluorocarbon gas of the formula C_(x)F_(y) intothe headspace; and subjecting the receptacle to high-shear mixing,thereby providing a suspension comprising microbubbles loaded with thebioactive gas.

Still other embodiments are directed to acoustically responsivemicrobubbles comprising a phospholipid monolayer shell, an encapsulatedbioactive gas selected from the group consisting of nitric oxide,hydrogen sulfide, and xenon gas, and an encapsulated perfluorocarbon gasof the formula C_(x)F_(y), wherein X is greater than or equal to 3, andwherein the volume ratio of bioactive gas to C_(x)F_(y) is from about10:1 to about 1:10.

All references (e.g., printed publications such as books, papers,patents, patent applications, catalogs, databases) are incorporatedherein by reference. In the event of a conflict or inconsistency, thepresent specification, as modified by any amendments thereto, shallcontrol.

These and other embodiments will be more clearly understood by referenceto the detailed disclosure and accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A schematic representation of the attenuation measurement setupis depicted. A pair of broadband PVDF transducers are employed toacquire the spectrum using a substitution technique. A pulser-receiverwas used in through-transmission mode to generate the excitation pulseand amplify the received signal. Samples of UCAs in diluent or diluentalone were added to the reservoir and introduced into the sample chamberby gravity feed.

FIG. 2. Attenuation spectra of (A) nitric oxide-loaded microbubbles, and(B) xenon-loaded microbubbles. In each case, microbubbles remainedattenuative even after 60 minutes, demonstrating stability of theformulations.

FIG. 3. Number-weighted size distribution of (A) nitric oxide-loadedmicrobubbles (NOMB), and (B) xenon-loaded microbubbles (XeMB). In eachcase, the microbubbles were small enough to pass through the capillariesof the lung.

FIG. 4. B-mode ultrasound image of a latex tube filled with (A) 0.5% BSAonly, and (B) 0.5% BSA with XeMB. The direction of flow is from left toright. The destruction of XeMB at the site of Doppler ultrasoundinsonation is apparent, showing the feasibility of ultrasound-mediatedgas delivery.

FIG. 5. Quantification of NO using an amperometric probe. Intact NOMBmeasurement denotes concentration of NO in the background. Broken NOMBmeasurement represents the overall measured concentration (NO that isencapsulated and dissolved in the background). (A) When 100% NO wasused, no difference was obtained between the NO concentration measuredwith intact microbubbles and broken microbubbles. (B) The concentrationof encapsulated NO was determined by subtracting background NOconcentration amount from the overall concentration (n=5).

FIG. 6. Attenuation spectroscopy measurements of the effluent obtainedafter insonification of XeMB.

FIG. 7. Attenuation spectrum of XeMB effluent injected through 19-gaugeand 25-gauge needles.

DETAILED DESCRIPTION

The clinical goal of liposomes designed for diagnostics and therapy isto deliver a pharmaceutical agent to the injured area. Liposomes aresubstantially spherical, self-assembling closed structures formed ofconcentric lipid bilayers with an aqueous phase inside and between thelipid bilayers. Their ability to entrap different water-solublecompounds within the inner aqueous phase and lipophilic agents betweenliposomal bilayers upon self-assembly has made them useful for deliveryof different kinds of drugs and for carrying diagnostic agents in avariety of imaging modalities. It is known that modification of theliposome shell with polyethylene glycol (PEG) enhances circulation timeand a common strategy is to attach antibodies or different bindingmoieties to the liposomal surface to target specific affected areas.Such modified liposomes are currently under investigation for targetedintravascular drug delivery to cells and noncellular components (such asendothelial cells, subendothelial structures, and blood components) asthe targeted sites for diagnosing and treating cardiac pathologies,including myocardial infarction, coronary thrombosis, andatherosclerosis.

Myocardial infarction (MI) results from occlusion of coronary arteriesby thrombi. During the ischemic phase and following reperfusion,extensive myocardial cell death occurs within the ischemic zone. The useof liposomes for delivery of MRI contrast agents and the use of PEG toincrease circulation time (substantially by avoiding recognition byliver cells), as well as the incorporation of binding partners such asantibody onto the liposome surface to achieve targeted delivery, are allstrategies known in the art. Visualization of thrombi and thrombolytictherapy are now mostly based on liposome-based targeted delivery ofcontrast agents and thrombolytic drugs, such as the enzymes urokinase,streptokinase, and tissue plasminogen activator (tPA).

A recent approach utilizes acoustically reflective (echogenic) liposomes(ELIP) that can be targeted to promote site-specific acousticenhancement of either imaging or drug delivery. Ultrasound-mediated drugdelivery is a relatively new technique for enhancing the penetration ofdrugs into diseased tissue beds noninvasively. By encapsulating drugsinto microsized and nanosized liposomes, the therapeutic can be shieldedfrom degradation within the vasculature until delivery to a target siteby ultrasound exposure. For example, Doppler ultrasound treatment hasbeen shown to result in earlier and more complete recanalization rateswhen tPA-loaded ELIP are co-administered. Echogenic liposomes have beenused to further develop the targeted delivery of tPA and to investigatethe effect of ultrasound exposure on thrombolytic efficacy. tPA isreleased from the nano-sized delivery complex when exposed toultrasound.

Previous studies have suggested that encapsulating nitric oxide (NO)with other gas components may improve the delivery profile of NO;however an effective bioactive mixture with sufficiently reduceddiffusion has yet to be designed. Perfluorocarbons, which are alreadyknown and approved as conventional ultrasound contrast agents due totheir low solubility in aqueous media and their low diffusivity comparedto low-molecular weight, biologically inert gases such as N₂, were aninitially thought to provide a possible solution, in particular since NOis soluble in certain perfluorocarbons such as octafluoropropane (OFP).Thus, the presence of OFP would theoretically delay diffusion of NO outof the liposome. However early studies concluded that the trade-off inlowered bioactivity versus preventing free diffusion was not desirable,as very large radius liposomes would be required in order to accommodatethe required encapsulation volume determined in the prior art.

The short half-life of bioactive gases such as NO due to hemoglobinscavenging has been an impediment to the therapeutic use of bioactivegases. Encapsulating a bioactive gas inside a lipid microbubble enablesthe gas to remain bioactive over a clinically relevant time frame. NO,for example, is a small molecule that can diffuse into surrounding fluidthrough a lipid bilayer or monolayer. Previous attempts to deliver NO tothe bloodstream have used a liquid perfluorocarbon emulsion, and not agas perfluorocarbon mixture (Rafikova, et al., Control of plasma nitricoxide bioactivity by perfluorocarbons: Physiological mechanisms andclinical implications, Mol. Card. 110: 3573-80 (2004). NO is also highlyreactive and degrades quickly in the presence of oxygen. Previousresearch has shown that use of a sparingly soluble trapped species cansignificantly enhance emulsion stability. While not desiring to be boundby theory, one explanation is Raoult's law, where the chemical potentialgradient for oxygen to dissolve into the microbubble in order to dilutethe osmotic agent balances the chemical potential gradient for oxygen todissolve out of the microbubble brought on by the capillary pressure(Taylor, Ostwald ripening in emulsions, Advances in Colloid andInterface Science. 75:107-63 (1998); Kabalnov, Ostwald Ripening andRelated Phenomena, Journal of Dispersion Science and Technology 22: 1-12(2001); Kwan, et al., Theranostic oxygen delivery using ultrasound andmicrobubbles, Theranostics 2: 1174-84 (2012)).

Octafluoropropane (OFP) belongs to a class of perfluorocarbons that areused alone as the gas phase in conventional ultrasound contrast agents,due to their low solubility in aqueous media and their low diffusivity.Combining NO with OFP enables a tradeoff between stability andtherapeutic dose. The instant inventors have previously reported thefeasibility of producing vasodilatation by delivery of NO into a viablecarotid artery during exposure to ultrasound. The formulation employed acombination of the following lipids to encapsulate NO:Dipalmitoylphosphatidylcholine (DPPC),N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP),polyethylene glycol (PEG) 2000, and PEG 750 in a 79:15:3:3 molar ratio.Experience with this formulation revealed that NO alone could not beloaded within the microbubbles formed by the lipid shell. It was foundthat a 1:1 volume ratio of NO and OFP was sufficient to permit robustacoustic response while retaining bioactivity commensurate withstandard-of-care vasodilators (Sutton et al. 2014). The instantinventors have now found that a different lipid formulation comprisingDSPC encapsulates orders of magnitude higher gas payload than theprevious DOTAP-containing formulation. The sequestering of nitric oxideby octafluoropropane, as well as the orders of magnitude increase inechogenicity and bioactive gas loading by changing the lipid constituentof the shell (without changing the lipid concentration) was notpredicted. The same approach was also extended to produce Xe-loadedmicrobubbles for cerebrovascular applications.

As used herein, the term “acoustically responsive” refers tomicrobubbles that are echogenic, which refers to contrast on a B-modeultrasound image, but which also vibrate in response to an acousticwave. It is this vibration that triggers the release of encapsulatedbioactive gas from the microbubble.

Microbubbles and Compositions

The present inventors have found that a perfluorocarbon gas as a trappedspecies or osmotic agent increases the stability of the microbubbleswithout significantly affecting the loading capacity. Surprisingly, itwas also found that presence of the perfluorocarbon prevents diffusionof oxygen into the microbubbles, thereby reducing degradation ofbioactive gas in situ—for example, the degradation of NO into nitrogendioxide, NO₂, a pollutant. Combining a bioactive gas, such as NO,hydrogen sulfide, or xenon, with a perfluorocarbon gas, such as OFP,unexpectedly sequesters the bioactive gas within the phospholipidmonolayer, thereby reducing its free diffusion into the surroundingmedia and preventing the corruption of the bioactive gas by oxygendiffusing into the microbubble.

Thus, certain embodiments provide an acoustically responsive microbubblecomprising a phospholipid monolayer shell, an encapsulated bioactive gasselected from the group consisting of nitric oxide, hydrogen sulfide,and xenon gas, and an encapsulated perfluorocarbon gas of the formulaC_(x)F_(y), wherein X is greater than or equal to 3, and wherein thevolume ratio of bioactive gas to C_(x)F_(y) is from about 10:1 to about1:10, including all values in the range. In a very specific embodiment,the ratio of bioactive gas to C_(x)F_(y) is about 10:1.

In a specific embodiment, the perfluorocarbon gas is OFP, wherein X is 3and Y is 8. However, the skilled artisan will appreciate that variousperfluorocarbon gases are suitable for use in the present compositionsand methods, including but not limited to perfluoropropane (C₃F₈),perfluorobutane (C₄F₁₀), perfluorocyclobutane (C₄F₈), perfluoropentane(C₅F₁₂), perfluorocyclopentane (C₅F₁₀), perfluoromethylcyclobutane(C₅F₁₀), perfluorohexane (C₆F₄), perfluorocyclohexane (C₆F₁₂),perfluoromethylcyclopentane (C₆F₁₂), perfluorodimethylcyclobutane(C₆F₁₂), perfluoroheptane (C₇F₁₆), perfluorocycloheptane (C₇F₁₄),perfluoromethylcyclohexane (C₇F₁₄), perfluorodimethylcyclopentane(C₇F₁₄), perfluorotrimethylcyclobutane (C₇F₁₄), and the like.

A variety of bioactive gases may be encapsulated in the presentlydisclosed microbubbles. In certain embodiments, the term “bioactive gas”refers to a bioactive gas selected from the group consisting of nitricoxide (NO), xenon (Xe), hydrogen sulfide (H₂S).

In some embodiments, the phospholipid monolayer shell comprises1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG 2000). The phospholipid monolayer in someembodiments is at least partially pegylated.

In other embodiments, it is desirable to incorporate target moietiesinto the phospholipid monolayer shell, in order to direct accumulationof the microbubbles at the target diseased section of vasculature. Insuch embodiments, the phospholipid monolayer shell is engineered tocontain molecules that adhere to cells, which express disease-specificmarkers (e.g., amino acids) on the membrane. In this way, themicrobubbles disclosed herein can be targeted to molecular components ofdisease by attaching specific target moieties to the surface of thephospholipid shell. Suitable target moieties include, but are notlimited to, small-molecule ligands, peptides, proteins, and monoclonalantibodies.

For purposes of treating a subject in need of promoting vasodilation,acoustically responsive microbubbles are formulated into an intra venous(IV) composition and injected or otherwise administered to the subjectat a site remote from the target treatment area, for example diseasedcardiovascular tissue. In one specific embodiment, the target treatmentarea is monitored for presence of the acoustically responsivemicrobubbles and upon detection of presence, acoustic energy is appliedsufficient to cause stable inertial or non-inertial cavitation dependingon the clinical goal.

Preparation of liposomes or microbubbles into pharmaceutical-gradecompositions formulated for IV administration is known in the art. Inparticular, Toh et al., Liposomes as sterile preparations andlimitations of sterilization techniques in liposomal manufacturing,Asian Journal of Pharmaceutical Sciences, 8(2):88-95 (April 2013)provides guidance for formulation of IV compositions of liposomes of thesizes of the microbubbles disclosed herein and provides useful guidancefor formulation of compositions of the presently described microbubbles.The entire disclosure of Toh et al. is incorporated herein by thisreference.

Methods of Promoting Vasodilation

In one embodiment a method for promoting vasodilation in a patient inneed thereof is provided, the method comprising: delivering amicrobubble comprising a phospholipid monolayer shell and anencapsulated bioactive gas locally to a target diseased section of thepatient's vasculature; and releasing the bioactive gas at the targetdiseased section, wherein the microbubble comprises the bioactive gas ina volume ratio of from about 10:1 to about 1:10 with a perfluorocarbongas. In a more specific embodiment, the volume ratio of bioactive gas toperfluorocarbon gas is about 10:1.

A variety of bioactive gases may be encapsulated in the presentlydisclosed microbubbles. In certain embodiments, the term “bioactive gas”refers to a bioactive gas selected from the group consisting of nitricoxide (NO), xenon (Xe), hydrogen sulfide (H₂S).

In some embodiments, delivering locally comprises administering themicrobubble comprising the bioactive gas to the patient at a site remotefrom the target diseased section; monitoring for presence of theadministered microbubble at the target diseased section; and upondetection of presence, administering acoustic energy to the targetdiseased section, thereby releasing a therapeutically effective amountof the bioactive gas from the microbubble to the target diseased sectionfor a clinically relevant time frame.

The acoustic energy may be provided as continuous or pulsed waveforms.In specific embodiments the ultrasound is pulsed. In other more specificembodiments, rest periods wherein no ultrasound is delivered to thetarget area for a measurable amount of time may be incorporated intoeither continuous or pulsed ultrasound delivery. The rest periods may bepatterned or sporadic. Such rest periods may provide opportunity forreperfusion of the target treatment area.

In certain embodiments, the perfluorocarbon gas has the formulaC_(x)F_(y) and X is greater than or equal to three. In a specificembodiment, the perfluorocarbon gas is OFP, wherein X is 3 and Y is 8.

In a specific embodiment, the phospholipid monolayer shell is at leastpartially pegylated and comprises1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG 2000).

In other embodiments, it is desirable to incorporate target moietiesinto the phospholipid monolayer shell, in order to direct accumulationof the microbubbles at the target diseased section of vasculature. Insuch embodiments, the phospholipid monolayer shell is engineered tocontain molecules that adhere to cells, which express disease-specificmarkers (e.g., amino acids) on the membrane. In this way, themicrobubbles disclosed herein can be targeted to molecular components ofdisease by attaching specific target moieties to the surface of thephospholipid shell. Suitable target moieties include, but are notlimited to, small-molecule ligands, peptides, proteins, and monoclonalantibodies.

As noted above, the present methods are useful for promotingvasodilation in a patient in need thereof. Various cardiovasculardiseases and conditions benefit from therapeutic treatment to promotevasodilation, including but not limited to, promotion of vasodilation inthe brain or the heart, reduction of vasospasm in post traumatic braininjury, treatment of bacterial endocarditis, treatment of biofilm growthon an indwelling catheter or internal body surface, promotion ofneuroprotection and reduction of reperfusion injury in the treatment ofstroke, and promotion of cardioprotection and reduction of reperfusioninjury in the treatment of myocardial infarction. Promoting vasodilationmay also reduce the risk of certain other undesirable medicalconditions. In some embodiments, the antiproliferative effects of NOreduce risk of neointimal hyperplasia post stent deployment or within anarteriovenous fistula for dialysis access.

According to another embodiment, a method for preventing passivediffusion of a bioactive gas from a microbubble into a non-target tissueor fluid is provided, the method comprising encapsulating the bioactivegas with a perfluorocarbon gas of the formula C_(x)F_(y), wherein X isgreater than or equal to three and wherein a volume ratio of bioactivegas to perfluorocarbon gas is from about 10:1 to about 1:10.

Preparation of a Microbubble Suspension

In specific manufacturing embodiments, the methods comprise: providing alipid dispersion comprising phospholipids in a sealed receptacle such asa glass vial, evacuating air from a headspace of the sealed receptacle,injecting a volume ratio of a bioactive gas and a perfluorocarbon gas ofthe formula C_(x)F_(y) into the headspace; and subjecting the receptacleto high-shear mixing, thereby providing a suspension comprisingmicrobubbles loaded with the bioactive gas. In methods known in theprior art, freezing of a lipid emulsion was considered to be a necessarystep for achieving encapsulation of gases and/or other agents at desiredvolumes. According to embodiments provided herein, freezing of the lipiddispersion is utilized simply to remove the residual solvents duringlipid film preparation. Instead, high shear mixing is used to generatelipid-encapsulated microbubbles.

In certain embodiments, the volume ratio of bioactive gas toperfluorocarbon gas is from about 10:1 to about 1:10. In very specificembodiments, X is 3, F is 8 (“OFP”), and the ratio of bioactive gas:OFPis 10:1.

According to specific embodiments, the lipid dispersion comprises amixture of phospholipids. According to very specific embodiments, thelipid dispersion formulation comprises: DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine) and DSPE-PEG 2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] dispersed in a solution containing phosphate bufferedsaline:propylene glycol:glycerol in a molar ratio of 16:3:1, with afinal lipid concentration of 1 mg/mL. As shown in the examples,stability of this formulation was demonstrated at physiologictemperature. It was found that microbubbles comprising DSPC encapsulateorders of magnitude higher gas payload than formulations comprisingDOTAP. The sequestering of NO gas by OFP gas, as well as the orders ofmagnitude increase in acoustic responsiveness and bioactive gas loadingobserved in microbubbles comprising DSPC without changing the lipidconcentration was unexpected. Similar results were observed inxenon-loaded microbubbles for cerebrovascular applications.

Quantifying Bioactive Gas Load

Also provided herein are methods for quantifying an amount of nitricoxide gas encapsulated within microbubbles in a suspension, the methodcomprising: providing a known volume of a suspension comprisingmicrobubbles encapsulating nitric oxide; transferring the suspensioninto degassed phosphate buffered saline; stirring the suspensioncontinuously to release the nitric oxide from the microbubbles into thephosphate buffered saline; measuring the nitric oxide dissolved in thephosphate buffered saline to determine total nitric oxide in thesuspension; and comparing the total nitric oxide in the suspension to acontrol value to quantify the amount of nitric oxide encapsulated withinthe microbubbles of the suspension.

Methods for quantifying an amount of xenon encapsulated withinmicrobubbles in a suspension are also provided, the method comprising:providing a known volume of a suspension comprising microbubblesencapsulating xenon in a sealed receptacle such as a glass vial;sonicating the receptacle; measuring the amount of xenon in a headspaceof the receptacle to determine total xenon in the suspension; comparingthe total xenon in the suspension to a control value to quantify theamount of xenon encapsulated within the microbubbles of the suspension.In a specific embodiment, the amount of xenon in the headspace of thereceptacle is measured by gas chromatography-mass spectrometry (GC-MS).

The following Examples are set forth to illustrate particularembodiments of the invention and should not be construed as limiting thefull scope of the invention as defined by the claims and understood by aperson of skill in the art.

EXAMPLES Example 1 Preparation of Bioactive Gas-EncapsulatingMicrobubbles

Bioactive gas-loaded microbubble agents were developed thatencapsulated 1) Nitric Oxide (NOMB), and 2) Xenon (XeMB). These agentswere encapsulated by a phospholipid shell with a PEG coating. Briefly,DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and DSPE-PEG 2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] were dissolved at a molar ratio of 9:1 chloroform and thesolution was evaporated to form a thin film. The thin-film was driedovernight under vacuum using a lyophilizer. The film was rehydratedusing a solution containing phosphate buffered saline:propyleneglycol:glycerol (16:3:1), which resulted in a final lipid concentrationof 1 mg/mL. This solution was sonicated in a bath sonicator for 45minutes to obtain clear lipid dispersion. Glass vials (2-mL volume) werealiquoted with 1 mL of lipid dispersion, sealed using Teflon/ptfe capsand the headspace was evacuated using a laboratory vacuum. Either NitricOxide or Xenon (1 mL) was injected into the headspace of each vial. Thevials were activated by high-shear mixing for 45 s using a Vialmix(Lantheus, N. Billerica, Mass., USA), which produced in a cloudysuspension containing microbubbles loaded with either bioactive gas.

Example 2 Attenuation Spectroscopy

Attenuation spectroscopy was used to confirm the encapsulation of gaswithin the microbubbles formulation, as well as assess the stability ofgas-encapsulated microbubbles over time. Set-up is depicted in FIG. 1.The frequency-dependent attenuation coefficient was measured using abroadband substitution technique as described previously (Raymond et al2014). After the gas-loaded microbubbles were manufactured, they werediluted in 0.5% (w/v) bovine serum albumin (a blood-mimicking fluid) andtheir attenuation spectrum over the frequency range 2-25 MHz wasmeasured over 60 minutes. The microbubbles continued to attenuateultrasound over 60 minutes, which highlights the ability of the presentformulation to keep bioactive gas sequestered for a clinically relevanttime frame. FIG. 2 shows attenuation spectra of (A) nitric oxide-loadedmicrobubbles, and (B) xenon-loaded microbubbles. In each case,microbubbles remained attenuative even after 60 minutes, demonstratingstability of the formulations.

Example 3 Size Distribution Assessment

Bioactive gas-loaded microbubbles were diluted in phosphate bufferedsaline and their size distribution was measured using a Coulter counter(Multisizer 4, Beckman Coulter, Brea, Calif., USA) to producenumber-weighted size distributions. For both formulations (NOMB andXeMB), over 99.9% of microbubbles were smaller than 7 μm (FIG. 3), whichwill facilitate passage through the capillaries of the lung and preventmicroemboli. FIG. 3 shows number-weighted size distribution of (A)nitric oxide-loaded microbubbles (NOMB), and (B) xenon-loadedmicrobubbles (XeMB). In each case, the microbubbles were small enough topass through the capillaries of the lung.

Example 4 Feasibility of Ultrasound-Mediated Release

To assess the feasibility of ultrasound-mediated release, XeMB wereexposed to Doppler Ultrasound pulses (center frequency=6 MHz, mechanicalindex=0.53, sample volume=1 mL). FIG. 4 shows a B-mode ultrasound imageof the longitudinal section of a latex tube perfused with 0.5% BSA in awater tank. The lumen on the tube is hypoechoic. However, when XeMB wasperfused into the system, the lumen became hyperechoic due to scatteringfrom the Xenon-loaded microbubbles. The microbubbles were destroyed uponexposure to Doppler ultrasound, which demonstrated the feasibility ofultrasound-mediated release of bioactive gases

Example 5 Quantification of Nitric Oxide Encapsulated within NitricOxide-Loaded Microbubbles (NOMB)

The amount of NO encapsulated within NOMB was measured using acalibrated amperometric probe (TBR4100, World Precision Instruments,Sarasota, Fla.). Specifically, a known volume of NOMB suspension wastransferred into degassed phosphate buffered saline (PBS) held in aglass container at 37° C. The PBS was either air saturated or degassed(pO₂=40%), and stirred continuously using a magnetic stirrer.Microbubbles are known to be stable in air-saturated PBS. On thecontrary, degassed PBS is known to force the diffusion of NO from NOMBinto the PBS. The concentration of dissolved NO in solution was measuredusing the calibrated probe. This measurement provided the totalconcentration of NO in the NOMB suspension. To measure the amount to NOthat is encapsulated in microbubbles, a subtraction-based approach wasfollowed. Specifically, the concentration of NO was measured with theNOMB diluted in air-saturated PBS (See FIG. 5, Intact NOMB). The amountof NO measured was considered as background. The amount of NOencapsulated was calculated by subtracting the background from the totalconcentration of NO measured by breaking NOMB.

To test the role of octafluoropropane (OFP) in stabilizing NOMBosmotically, NOMB were prepared by injecting 1 ml of gas (either 100% NOor 90% NO and 10% OFP (v/v)) in the vial headspace followed byhigh-shear mixing. When NOMB were prepared with 100% NO gas, nodifference was found between the background and total NO measurement(FIG. 5(A)), likely because all NO diffused out almost instantaneouslyfrom the microbubble into the solution. However, when NOMB were preparedby adding the NO/OFP gas mixture, the amount of NO encapsulated withinNOMB was found to be nearly 0.7 mM. These results show that adding asmall percentage of OFP by volume is critical to encapsulating NO withinNOMB (FIG. 5(B)).

Example 6 Quantification of Xenon Encapsulated Inside XeMB

A known volume of Xenon encapsulated microbubbles (XeMB) was transferredto a capped glass vial. The vial was submerged in a bath and sonicatedat 40-kHz frequency for 5 min to release the entire payload of Xe fromXeMB. The amount of Xe released was measured using headspace analysiswith a mass spectrometer (5973A, The Hewlett-Packard Company) interfacedwith a gas chromatograph (Model 6890, Hewlett-Packard Company). Thedifference in Xe concentration measured with and without sonication wasused to quantify the volume of gas encapsulated per milligram of lipidwithin XeMB. The results revealed that XeMB encapsulated 800/mg of xenonper milligram of lipid, which is at least four-fold higher than thepreviously reported xenon-loaded echogenic liposome formulations.

Example 7 Ultrasound-Mediated Release of Xe from XeMB

Assessment of the acoustic response of XeMB is important forultrasound-mediated release of xenon. The acoustic response of XeMB wasevaluated using an attenuation spectrometer. Specifically, the XeMBsuspension was diluted 1000-fold using 0.5% bovine serum albumin andallowed to flow into a Clinicell® (CLINIcell® 25, Mabio, Tourcoing,France) through a latex tube (5 mm diameter) at a flow rate of 5 ml/min.The latex tube was exposed to ultrasound using a commercial ultrasoundsystem (HDI 5000, Philips). Three insonation different modes were testedon XeMB individually and attenuation spectroscopy studies were performedwith the effluent. FIG. 6 shows the attenuation coefficients of theeffluents collected after XeMB were insonified under three differentmodes (B-mode, Color Doppler, and Pulsed Doppler). B-mode imaging wasperformed at a very low Mechanical Index (0.03) as a negative controlfor Xe release. Pulsed and color Doppler were used at mechanical indicesof 0.53 and 0.49, respectively to try to induce the release of Xe fromXeMB. The sample volume used for pulsed Doppler ultrasound was 1 ml. Inresponse to both Doppler ultrasound modes, the attenuation coefficientdropped by almost 20-30 dB, which suggests that Xe was released fromXeMB.

Example 8 Stability of XeMB when Injected Through 19-Guage and 25-GaugeNeedles

Stability of XeMB was studied when injected through a 19-gauge and25-gauge needle at a flow-rate of 4 ml/min and 0.2 ml/min, respectively.These flow rates mimic the flow rates used clinically and in preclinicalstudies for ultrasound contrast agents. The effluent was collected andthe attenuation coefficient of the suspension measured. The resultsrevealed that the XeMB attenuation coefficient measured after injectionthrough a 25-gauge needle was within one standard deviation of thatmeasured using a 19-gauge needle. See FIG. 7. These results suggest thatXeMB can be injected using either a 19-gauge or a 25-gauge needle basedon the intended application.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims. It will be appreciatedthat the invention is in no way dependent upon particular resultsachieved in any specific example or with any specific embodiment.Articles such as “a”, “an” and “the” may mean one or more than oneunless indicated to the contrary or otherwise evident from the context.Claims or descriptions that include “or” between one or more members ofa group are considered satisfied if one, more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process unless indicated to the contrary or otherwiseevident from the context. The invention includes embodiments in whichexactly one member of the group is present in, employed in, or otherwiserelevant to a given product or process. The invention also includesembodiments in which more than one, or all group members are present in,employed in, or otherwise relevant to a given product or process.Furthermore, it is to be understood that the invention encompasses allvariations, combinations, and permutations in which one or morelimitations, elements, clauses, descriptive terms, etc., from one ormore of the listed claims or from the description above is introducedinto another claim. For example, any claim that is dependent on anotherclaim can be modified to include one or more elements, limitations,clauses, or descriptive terms, found in any other claim that isdependent on the same base claim. Furthermore, where the claims recite acomposition, it is to be understood that methods of using thecomposition for any of the purposes disclosed herein are included withinthe scope of the invention, and methods of making the compositionaccording to any of the methods of making disclosed herein are includedwithin the scope of the invention, unless otherwise indicated or unlessit would be evident to one of ordinary skill in the art that acontradiction or inconsistency would arise. Methods can include a stepof providing a subject suffering from a disease or condition thatbenefits from therapeutically promoting vasodilation, a step ofdiagnosing a subject as having a disease or condition that benefits fromtherapeutically promoting vasodilation (such as cardiovascular disease),and/or a step of selecting a subject for which an inventive product ormethod would be suitable.

Where elements are presented as lists, it is to be understood that eachsubgroup of the elements is also disclosed, and any element(s) can beremoved from the group. For purposes of conciseness only some of theseembodiments have been specifically recited herein, but the inventionincludes all such embodiments. It should also be understood that, ingeneral, where the invention, or aspects of the invention, is/arereferred to as comprising particular elements, features, etc., certainembodiments of the invention or aspects of the invention consist, orconsist essentially of, such elements, features, etc.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise. Any particular embodiment, aspect,element, feature, etc., of the present invention, or any combinationthereof, may be explicitly excluded from any one or more claims whetheror not such exclusion is expressly recited herein. For example, anylipid shell or encapsulated gas component ingredient, etc., can beexplicitly excluded. Applicants reserve the right to proviso out of theclaims any specific component, component category, or combinationthereof, whether or not such component, category, or combinationthereof, is recited herein. To the extent, if any, that a echogenicliposome that is known or described in the prior art may include nitricoxide, the instant invention may be distinguished from such prior artliposome or methods utilizing such prior art liposome in, for example,any one or more of the following ways: (i) the acoustically responsivemicrobubbles of the invention comprises one or more gases or lipid shellcomponents or configurations (e.g., monolayer phospholipid shell) notpresent in the prior art liposome or microbubble; (ii) the acousticallyresponsive microbubble of the invention comprises a different amount ofspecific components, or a specific ratio of specific components, notrecognized or appreciated as significant to utility or efficacy in theprior art liposome or microbubble and methods; or (iii) the acousticallyresponsive microbubble or methods of the invention omit at least oneingredient present and considered necessary in the prior art liposome ormicrobubble or methods.

What is claimed:
 1. A method for treating a condition that benefits frompromoting vasodilation in a patient in need thereof, the methodcomprising: delivering a microbubble comprising a phospholipid monolayershell and an encapsulated therapeutic bioactive gas locally to a targetdiseased section of the patient's vasculature; and releasing thetherapeutic bioactive gas at the target diseased section, wherein themicrobubble comprises the therapeutic bioactive gas in a volume ratio offrom about 10:1 to about 1:10 with a perfluorocarbon gas.
 2. The methodaccording to claim 1, wherein the ratio is about 10:1.
 3. The methodaccording to claim 1, wherein the therapeutic bioactive gas is selectedfrom the group consisting of nitric oxide, xenon, and hydrogen sulfide.4. The method according to claim 3, wherein antiproliferative effects ofnitric oxide reduce risk of neointimal hyperplasia post stent deploymentor promote patency in an arteriovenous fistula for dialysis access. 5.The method according to claim 1, wherein delivering locally comprisesadministering the microbubble comprising the therapeutic bioactive gasto the patient at a site remote from the target diseased section; andadministering acoustic energy to the target diseased section, therebyreleasing a therapeutically effective amount of the therapeuticbioactive gas from the microbubble to the target diseased section for aclinically relevant time frame.
 6. The method according to claim 5,further comprising monitoring for presence of the administeredmicrobubble at the target diseased section prior to administeringacoustic energy to the target diseased section, wherein acoustic energyis administered upon detection of presence of the administeredmicrobubble.
 7. The method according to claim 1, wherein theperfluorocarbon gas has the formula C_(x)F_(y) and X is greater than orequal to three.
 8. The method according to claim 7, wherein X=3 and Y=8.9. The method according to claim 1, wherein the phospholipid monolayershell is pegylated.
 10. The method according to claim 1, wherein thephospholipid monolayer shell further comprises target moieties fordirecting accumulation of the microbubbles at the target diseasedsection.
 11. The method according to claim 1, wherein the condition isselected from the group consisting of cardiovascular disease,cerebrovascular disease, vasospasm post traumatic brain injury,bacterial endocarditis, biofilm growth on an indwelling catheter orinternal body surface, and maintenance of arteriovenous fistula fordialysis access.
 12. A method of treating cardiovascular disease in apatient in need thereof, the method comprising: delivering a microbubblecomprising a phospholipid monolayer shell and an encapsulated bioactivegas locally to a target diseased section of the patient's vasculature;and releasing the bioactive gas at the target diseased section, whereinthe microbubble comprises the bioactive gas in a volume ratio of fromabout 10:1 to about 1:10 with a perfluorocarbon gas, wherein theperfluorocarbon gas has the formula C_(x)F_(y) and X is greater than orequal to three.
 13. The method according to claim 12, wherein thebioactive gas is selected from the group consisting of nitric oxide,xenon, and hydrogen sulfide.
 14. The method according to claim 13,wherein delivering locally comprises administering the microbubblecomprising the bioactive gas to the patient at a site remote from thetarget diseased section; and administering acoustic energy to the targetdiseased section, thereby releasing a therapeutically effective amountof the bioactive gas from the microbubble to the target diseased sectionfor a clinically relevant time frame.
 15. The method according to claim14, further comprising monitoring for presence of the administeredmicrobubble at the target diseased section prior to administeringacoustic energy to the target diseased section, wherein acoustic energyis administered upon detection of presence of the administeredmicrobubble.
 16. The method according to claim 12, wherein X=3 and Y=8.17. The method according to claim 12, wherein the phospholipid monolayershell further comprises target moieties for directing accumulation ofthe microbubbles at the target diseased section.